DNase is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. DNase has been purified from various species to various degrees. The complete amino acid sequence for a mammalian DNase was first made available in 1973. See, e.g., Liao, et al., J. Biol. Chem. 248, 1489 (1973).
DNase has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions in such diseases as pneumonia and cystic fibrosis, thereby aiding in the clearing of respiratory airways. See, e.g., Lourenco, et al., Arch. Intern. Med. 142, 2299 (1982); Shak, et al., Proc. Nat. Acad. Sci. 87, 9188 (1990); and Hubbard, et al., New England Journal of Medicine 326, 812 (1992).
DNA encoding human DNase has been isolated and sequenced and that DNA has been expressed in recombinant mammalian host cells, thereby enabling the production of human DNase in mammalian commercially useful quantities. See, e.g., Shak, et al., Proc. Nat. Acad. Sci. 87, 9188 (1990). Recombinant human DNase (rhDNase) has been found to be useful clinically, especially in purified form such that the DNase is free from proteases and other proteins with which it is ordinarily associated in nature.
The means and methods by which human DNase can be obtained in pharmaceutically effective form is described in the patent applications cited above. Various specific methods for the purification of DNase are known in the art. See, e.g., Khouw, et al., U.S. Pat. No. 4,065,355, issued 27 Dec. 1977; Markey, FEBS Letters 167, 155 (1984); and Nefsky, et al., Euro. Journ. Biochem. 179, 215 (1989).
The present application is predicated on the use of such DNase for formulation. DNase can be employed as such, as a mixture of deamidated and non-deamidated forms, or in isolated deamidated and non-deamidated forms, The preparation and separation of such forms are the subject matter of a patent application cited above.
The present invention is directed to the preparation of liquid solutions of DNase (including all of its biologically active forms as previously noted) that are stable to thermally induced aggregation of DNase.
The present invention is directed to the preparation of stabilized liquid solutions of DNase (including all of its biologically active forms as previously noted). These liquid solutions containing DNase in substantially non-deamidated form are maintained at pHs of less than neutral in stable form such that precipitation of material does not occur to any substantial extent, and therefore, the solutions are in a clear form suitable for pharmaceutical administration. Such less than neutral pH levels result in a reduction of the rate of deamidation of the DNase principle during storage. At storage at elevated temperatures (upwards of 37° C.), such lower pH solutions result in precipitation products. The present invention, in an aspect, stabilizes such solutions from such precipitation.